PubMed Health⌕ Search

Biomedical subjects

U H von Andrian

Publications and source records attributed to U H von Andrian.

At least 19 recordsLinked to original sources
PubMed2003

Gene therapy to target dendritic cells from blood to lymph nodes.

Peripheral lymph nodes (PLN) are strategic microenvironments where antigen-presenting dendritic cells (DC), loaded with environmental antigens, and naive lymphocytes meet to initiate immune responses. The unique capacity of DC to induce primary immune responses has led to their use in clinical medicine; however, delivering DC to lymph nodes is problematic. Intravenously injected DC cannot access to PLN, while DC injected into tissue migrate inefficiently through lymphatics to PLN. We achieved DC targeting to T-cell areas of PLN by endowing DC with a novel receptor for peripheral node addressin (PNAd), an adhesion molecule present on the lymph node venular endothelium. This novel receptor is a chimeric E/L-selectin (ELS) that, we have previously shown, binds to PNAd. DC were genetically modified by retroviral transduction to express ELS. ELS expression was targeted to tips of microvilli, and mediated rolling of DC on PNAd both in vivo and in vitro. Such genetically engineered DC could extravasate directly from blood through the lymph node endothelium as opposed to nontransduced DC. This study provides evidence that the trafficking of DC can be modified using gene transfer technologies. More efficient delivery of DC to PLN should assist the development of improved vaccination strategies.

Adoptive Transfer↗
PubMed2001

Inflammatory chemokine transport and presentation in HEV: a remote control mechanism for monocyte recruitment to lymph nodes in inflamed tissues.

Interstitial fluid is constantly drained into lymph nodes (LNs) via afferent lymph vessels. This conduit enables monocyte-derived macrophages and dendritic cells to access LNs from peripheral tissues. We show that during inflammation in the skin, a second recruitment pathway is evoked that recruits large numbers of blood-borne monocytes to LNs via high endothelial venules (HEVs). Inhibition of monocyte chemoattractant protein (MCP)-1 blocked this inflammation-induced monocyte homing to LNs. MCP-1 mRNA in inflamed skin was over 100-fold upregulated and paralleled MCP-1 protein levels, whereas in draining LNs MCP-1 mRNA induction was much weaker and occurred only after a pronounced rise in MCP-1 protein. Thus, MCP-1 in draining LNs was primarily derived from inflamed skin. In MCP-1(-/-) mice, intracutaneously injected MCP-1 accumulated rapidly in the draining LNs where it enhanced monocyte recruitment. Intravital microscopy showed that skin-derived MCP-1 was transported via the lymph to the luminal surface of HEVs where it triggered integrin-dependent arrest of rolling monocytes. These findings demonstrate that inflamed peripheral tissues project their local chemokine profile to HEVs in draining LNs and thereby exert "remote control" over the composition of leukocyte populations that home to these organs from the blood.

Adoptive Transfer↗
PubMed2001

Migratory properties of naive, effector, and memory CD8(+) T cells.

It has been proposed that two different antigen-experienced T cell subsets may be distinguishable by their preferential ability to home to lymphoid organs (central memory cells) or nonlymphoid tissues (effector memory/effector cells). We have shown recently that murine antigen-primed CD8(+) T cells cultured in interleukin (IL)-15 (CD8(IL-15)) resemble central memory cells in phenotype and function. In contrast, primed CD8(+) T cells cultured in IL-2 (CD8(IL-2)) become cytotoxic effector cells. Here, the migratory behavior of these two subsets was investigated. Naive, CD8(IL-15) cells and, to a lesser degree, CD8(IL-2) cells localized to T cell areas in the spleen, but only naive and CD8(IL-15) cells homed to lymph nodes (LNs) and Peyer's patches. Intravital microscopy of peripheral LNs revealed that CD8(IL-15) cells, but not CD8(IL-2) cells, rolled and arrested in high endothelial venules (HEVs). Migration of CD8(IL-15) cells to LNs depended on L-selectin and required chemokines that bind CC chemokine receptor (CCR)7. Both antigen-experienced populations, but not naive T cells, responded to inflammatory chemokines and accumulated at sites of inflammation. However, CD8(IL-2) cells were 12 times more efficient in migrating to inflamed peritoneum than CD8(IL-15) cells. Furthermore, CD8(IL-15) cells proliferated rapidly upon reencounter with antigen at sites of inflammation. Thus, central memory-like CD8(IL-15) cells home avidly to lymphoid organs and moderately to sites of inflammation, where they mediate rapid recall responses, whereas CD8(IL-2) effector T cells accumulate in inflamed tissues, but are excluded from most lymphoid organs.

Animals↗
PubMed2001

L-selectin shedding is independent of its subsurface structures and topographic distribution.

L-selectin (CD62L), a lectin-like adhesion molecule, mediates lymphocyte homing and leukocyte accumulation at sites of inflammation. Its transmembrane (TM) and intracellular (IC) domains confer clustering of L-selectin on microvilli of resting leukocytes, which is important for L-selectin function. Following activation of protein kinase C (PKC) or calmodulin inhibition, the wild-type (WT) protein is rapidly cleaved in its membrane-proximal ectodomain. To examine whether L-selectin topography or TM/IC domains are involved in this shedding process, we used stable transfectants expressing WT L-selectin (on microvilli) or chimeric molecules consisting of the L-selectin ectodomain linked to the TM/IC domains of CD44 (excluded from microvilli) or CD31 (randomly distributed). PKC activation by PMA altered the cells' surface morphology, but did not induce a redistribution of L-selectin ectodomains. All cell lines shed ectodomains upon PMA activation in a dose-dependent fashion and with similar kinetics. Calmodulin inhibition by trifluoperazine induced shedding in both WT and chimera transfectants. At high trifluoperazine concentrations, shedding of WT L-selectin was significantly more pronounced than that of chimeric molecules. Regardless of the activating stimulus, shedding was blocked by a hydroxamate-based metalloprotease inhibitor, suggesting that ectodomain down-regulation occurred through proteolytic cleavage by identical protease(s). These results show that the recognition site(s) for PKC-induced L-selectin shedding is exclusively contained within the ectodomain; the nature of subsurface structures and surface topography are irrelevant. Shedding induced by calmodulin inhibition has two components: one requires the L-selectin TM/IC domain, and the other is independent of it.

Animals↗
PubMed2001

Reversibly locking a protein fold in an active conformation with a disulfide bond: integrin alphaL I domains with high affinity and antagonist activity in vivo.

The integrin alphaLbeta2 has three different domains in its headpiece that have been suggested to either bind ligand or to regulate ligand binding. One of these, the inserted or I domain, has a fold similar to that of small G proteins. The I domain of the alphaM and alpha2 subunits has been crystallized in both open and closed conformations; however, the alphaL I domain has been crystallized in only the closed conformation. We hypothesized that the alphaL domain also would have an open conformation, and that this would be the ligand binding conformation. Therefore, we introduced pairs of cysteine residues to form disulfides that would lock the alphaL I domain in either the open or closed conformation. Locking the I domain open resulted in a 9,000-fold increase in affinity to intercellular adhesion molecule-1 (ICAM-1), which was reversed by disulfide reduction. By contrast, the affinity of the locked closed conformer was similar to wild type. Binding completely depended on Mg(2+). Orders of affinity were ICAM-1 > ICAM-2 > ICAM-3. The k(on), k(off), and K(D) values for the locked open I domain were within 1.5-fold of values previously determined for the alphaLbeta2 complex, showing that the I domain is sufficient for full affinity binding to ICAM-1. The locked open I domain antagonized alphaLbeta2-dependent adhesion in vitro, lymphocyte homing in vivo, and firm adhesion but not rolling on high endothelial venules. The ability to reversibly lock a protein fold in an active conformation with dramatically increased affinity opens vistas in therapeutics and proteomics.

Antigens, CD↗
PubMed2001

The CCR7 ligand elc (CCL19) is transcytosed in high endothelial venules and mediates T cell recruitment.

Lymphocyte homing to secondary lymphoid tissue is defined by a multistep sequence of interactions between lymphocytes and endothelial cells in high endothelial venules (HEVs). After initial selectin-mediated tethering and rolling, firm adhesion of lymphocytes requires rapid upregulation of lymphocyte integrin adhesiveness. This step is mediated in part by the HEV-derived chemokine SLC (secondary lymphoid-tissue chemokine, or CCL21) that binds to the CC chemokine receptor (CCR)7 on lymphocytes. However, the CC chemokine ELC (Epstein-Barr virus-induced molecule 1 ligand chemokine, or CCL19) shares the same receptor, and ELC transcripts have been observed in the T cell areas of lymphoid organs. Here, we show that perivascular ELC is transcytosed to the luminal surfaces of HEVs and enables efficient T cell homing to lymph nodes. In situ hybridization on sections of human tonsil showed no ELC mRNA in HEVs, but immunostaining revealed ELC protein in cytoplasmic vesicles of HEV cells. Furthermore, ELC injected into the footpads of mice entered the draining lymph nodes and was presented by HEVs. Finally, intracutaneous injections of ELC in mice lacking functionally relevant ELC and SLC (plt/plt mice) restored T cell trafficking to draining lymph nodes as efficiently as SLC. We conclude that perivascular ELC is transcytosed to the luminal surfaces of HEVs and participates in CCR7-mediated triggering of lymphocyte arrest.

Animals↗
PubMed2001

The alpha(1,3)fucosyltransferases FucT-IV and FucT-VII exert collaborative control over selectin-dependent leukocyte recruitment and lymphocyte homing.

E-, P-, and L-selectin counterreceptor activities, leukocyte trafficking, and lymphocyte homing are controlled prominently but incompletely by alpha(1,3)fucosyltransferase FucT-VII-dependent fucosylation. Molecular determinants for FucT-VII-independent leukocyte trafficking are not defined, and evidence for contributions by or requirements for other FucTs in leukocyte recruitment is contradictory and incomplete. We show here that inflammation-dependent leukocyte recruitment retained in FucT-VII deficiency is extinguished in FucT-IV(-/-)/FucT-VII(-/-) mice. Double deficiency yields an extreme leukocytosis characterized by decreased neutrophil turnover and increased neutrophil production. FucT-IV also contributes to HEV-born L-selectin ligands, since lymphocyte homing retained in FucT-VII(-/-) mice is revoked in FucT-IV(-/-)/FucT-VII(-/-) mice. These observations reveal essential FucT-IV-dependent contributions to E-, P-, and L-selectin ligand synthesis and to the control of leukocyte recruitment and lymphocyte homing.

Animals↗
PubMed2001

Characterization of a mouse model for thrombomodulin deficiency.

Mutations in the gene encoding thrombomodulin (TM), a thrombin regulator, are suspected risk factors for venous and arterial thrombotic disease. We have previously described the generation of TM(Pro/Pro) mice carrying a TM gene mutation that disrupts the TM-dependent activation of protein C. Here, it is shown that inbred C57BL/6J TM(Pro/Pro) mice exhibit a hypercoagulable state and an increased susceptibility to thrombosis and sepsis. Platelet thrombus growth after FeCl(3)-induced acute endothelial injury was accelerated in mutant mice. Vascular stasis after permanent ligation of the carotid artery precipitated thrombosis in mutant but not in normal mice. Mutant mice showed increased mortality after exposure to high doses of endotoxin and demonstrated altered cytokine production in response to low-dose endotoxin. The severity of the hypercoagulable state and chronic microvascular thrombosis caused by the TM(Pro) mutation is profoundly influenced by mouse strain-specific genetic differences between C57BL/6 and 129SvPas mice. These data demonstrate that in mice, TM is a physiologically relevant regulator of platelet- and coagulation-driven large-vessel thrombosis and modifies the response to endotoxin-induced inflammation. The phenotypic penetrance of the TM(Pro) mutation is determined by as-yet-uncharacterized genetic modifiers of thrombosis other than TM.

Animals↗
PubMed2001

Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes.

The lineage relationship between short-lived effector T cells and long-lived memory cells is not fully understood. We have described T-GFP mice previously, in which naive and early activated T cells express GFP uniformly, whereas cells that have differentiated into effector cytotoxic T cells selectively lose GFP expression. Here we studied antigen-specific CD8 T cell differentiation using T-GFP mice crossed to the TCR transgenic (Tg) mice P14 (specific for the lymphocytic choriomeningitis virus glycoprotein peptide, gp33-41). After activation with antigenic peptide, P14XT-GFP CD8(+) T cells cultured in high-dose IL-2 developed into cells with effector phenotype and function: they were blastoid, lost GFP expression, expressed high levels of activation and effector markers, and were capable of immediate cytotoxic function. In contrast, cells cultured in IL-15 or low-dose IL-2 never developed into full-fledged effector cells. Rather, they resembled memory cells: they were smaller, were GFP(+), did not express effector markers, and were incapable of immediate cytotoxicity. However, they mediated rapid-recall responses in vitro. After adoptive transfer, they survived in vivo for at least 10 weeks and mounted a secondary immune response after antigen rechallenge that was as potent as endogenously generated memory cells. In addition to providing a simple means to generate memory cells in virtually unlimited numbers, our results suggest that effector differentiation is not a prerequisite for memory cell generation.

Animals↗
PubMed2000

BLTR mediates leukotriene B(4)-induced chemotaxis and adhesion and plays a dominant role in eosinophil accumulation in a murine model of peritonitis.

Leukotriene B(4) (LTB(4)) is a potent chemoattractant active on multiple leukocytes, including neutrophils, macrophages, and eosinophils, and is implicated in the pathogenesis of a variety of inflammatory processes. A seven transmembrane-spanning, G protein-coupled receptor, called BLTR (LTB(4) receptor), has recently been identified as an LTB(4) receptor. To determine if BLTR is the sole receptor mediating LTB(4)-induced leukocyte activation and to determine the role of LTB(4) and BLTR in regulating leukocyte function in inflammation in vivo, we generated a BLTR-deficient mouse by targeted gene disruption. This mouse reveals that BLTR alone is responsible for LTB(4)-mediated leukocyte calcium flux, chemotaxis, and firm adhesion to endothelium in vivo. Furthermore, despite the apparent functional redundancy with other chemoattractant-receptor pairs in vitro, LTB(4) and BLTR play an important role in the recruitment and/or retention of leukocytes, particularly eosinophils, to the inflamed peritoneum in vivo. These studies demonstrate that BLTR is the key receptor that mediates LTB(4)-induced leukocyte activation and establishes a model to decipher the functional roles of BLTR and LTB(4) in vivo.

Animals↗
PubMed2000

The CC chemokine thymus-derived chemotactic agent 4 (TCA-4, secondary lymphoid tissue chemokine, 6Ckine, exodus-2) triggers lymphocyte function-associated antigen 1-mediated arrest of rolling T lymphocytes in peripheral lymph node high endothelial venules.

T cell homing to peripheral lymph nodes (PLNs) is defined by a multistep sequence of interactions between lymphocytes and endothelial cells in high endothelial venules (HEVs). After initial tethering and rolling via L-selectin, firm adhesion of T cells requires rapid upregulation of lymphocyte function-associated antigen 1 (LFA-1) adhesiveness by a previously unknown pathway that activates a Galpha(i)-linked receptor. Here, we used intravital microscopy of murine PLNs to study the role of thymus-derived chemotactic agent (TCA)-4 (secondary lymphoid tissue chemokine, 6Ckine, Exodus-2) in homing of adoptively transferred T cells from T-GFP mice, a transgenic strain that expresses green fluorescent protein (GFP) selectively in naive T lymphocytes (T(GFP) cells). TCA-4 was constitutively presented on the luminal surface of HEVs, where it was required for LFA-1 activation on rolling T(GFP) cells. Desensitization of the TCA-4 receptor, CC chemokine receptor 7 (CCR7), blocked T(GFP) cell adherence in wild-type HEVs, whereas desensitization to stromal cell-derived factor (SDF)-1alpha (the ligand for CXC chemokine receptor 4 [CXCR4]) did not affect T(GFP) cell behavior. TCA-4 protein was not detected on the luminal surface of PLN HEVs in plt/plt mice, which have a congenital defect in T cell homing to PLNs. Accordingly, T(GFP) cells rolled but did not arrest in plt/plt HEVs. When TCA-4 was injected intracutaneously into plt/plt mice, the chemokine entered afferent lymph vessels and accumulated in draining PLNs. 2 h after intracutaneous injection, luminal presentation of TCA-4 was detectable in a subset of HEVs, and LFA-1-mediated T(GFP) cell adhesion was restored in these vessels. We conclude that TCA-4 is both required and sufficient for LFA-1 activation on rolling T cells in PLN HEVs. This study also highlights a hitherto undocumented role for chemokines contained in afferent lymph, which may modulate leukocyte recruitment in draining PLNs.

Animals↗
PubMed2000

Specialized contributions by alpha(1,3)-fucosyltransferase-IV and FucT-VII during leukocyte rolling in dermal microvessels.

Noninflamed skin venules support constitutive leukocyte rolling. P-selectin controls the rolling frequency, whereas E-selectin dictates rolling velocity (Vroll). Fucosylated selectin ligands are essential for all interactions, as rolling was absent in mice doubly deficient in alpha1,3-fucosyltransferase (FucT)-IV and FucT-VII. The rolling fraction was reduced in FucT-VII-/- animals but normal in FucT-IV-/- mice. However, Vroll was markedly increased in both strains. P-selectin ligands generated by FucT-VII are crucial for initial leukocyte tethering, whereas E-selectin ligands that permit maximum slowing of Vroll require simultaneous expression of FucT-IV and FucT-VII. These results demonstrate a role for FucT-IV in selectin-dependent adhesion and suggest that the endothelial selectins and FucTs have distinct but overlapping functions in the immunosurveillance of the skin.

Animals↗
PubMed1999

The BCR/ABL oncogene alters the chemotactic response to stromal-derived factor-1alpha.

The chemokine stromal-derived factor-1alpha (SDF-1alpha) is a chemoattractant for CD34(+) progenitor cells, in vitro and in vivo. The receptor for SDF-1alpha, CXCR-4, is a 7 transmembrane domain receptor, which is also a coreceptor for human immunodeficiency virus (HIV). Here we show that transformation of hematopoietic cell lines by BCR/ABL significantly impairs their response to SDF-1alpha. Three different hematopoietic cell lines, Ba/F3, 32Dcl3, and Mo7e, were found to express CXCR-4 and to respond to SDF-1alpha with increased migration in a transwell assay. In contrast, after transformation by the BCR/ABL oncogene, the chemotactic response to SDF-1alpha was reduced in all 3 lines. This effect was directly due to BCR/ABL, because Ba/F3 cells, in which the expression of BCR/ABL could be regulated by a tetracycline-inducible promoter, also had reduced chemotaxis to SDF-1alpha when BCR/ABL was induced. The reduced response to SDF-1alpha was not due to an inability of BCR/ABL-transformed cell lines to migrate in general, as spontaneous motility of BCR/ABL-transformed cells was increased. In mice, injection of SDF-1alpha into the spleen resulted in a transient accumulation of untransformed Ba/F3 cells, but not Ba/F3. p210(BCR/ABL) cells administered simultaneously. The mechanism may involve inhibition of CXCR-4 receptor function, because in BCR/ABL-transformed cells, CXCR-4 receptors were expressed on the cell surface, but SDF-1alpha calcium flux was inhibited. Because SDF-1alpha and CXCR-4 are felt to be involved in progenitor cell homing to marrow, the abnormality decribed here could contribute to the homing and retention defects typical of immature myeloid cells in chronic myelogenous leukemia.

Animals↗
PubMed1999

A transgenic mouse model to analyze CD8(+) effector T cell differentiation in vivo.

Antigen-specific effector T cells are prerequisite to immune protection, but because of the lack of effector cell-specific markers, their generation and differentiation has been difficult to study. We report that effector cells are highly enriched in a T cell subset that can be specifically identified in transgenic (T-GFP) mice expressing green fluorescent protein (GFP) under control of the murine CD4 promoter and proximal enhancer. Consistent with previous studies of these transcriptional control elements, GFP was strongly and specifically expressed in nearly all resting and short-term activated CD4(+) and CD8(+) T cells. However, when T-GFP mice were challenged with vaccinia virus, allogeneic tumor cells, or staphylococcal enterotoxin A, the cytotoxic and IFN-gamma-producing T cells lost GFP expression. Upon T cell receptor (TCR) ligation by alphaCD3, sorted GFP(+) cells fluxed calcium and proliferated vigorously. In contrast, GFP(-) effector cells showed a diminished calcium flux and did not proliferate. Instead, they underwent apoptosis unless supplied with exogenous IL-2. By reverse transcription-PCR analysis, the GFP(-) cells up-regulated the pro-apoptotic molecule, Fas-L, and down-regulated gene expression of the proximal TCR signaling molecule, CD3zeta, and c-jun, a component of the AP-1 transcription factor. Thus, differential regulation of TCR signaling may explain the divergent responses of naïve and effector T cells to antigen stimulation.

Animals↗
PubMed1999

Interaction of dendritic cells with skin endothelium: A new perspective on immunosurveillance.

The goal of this study was to determine the mechanisms by which dendritic cells (DCs) in blood could interact with endothelium, a prerequisite to extravasation into tissues. Our results indicate that DCs express both HECA-452-reactive and nonreactive isoforms of P-selectin glycoprotein ligand 1 (PSGL-1) and can tether and roll efficiently on E- and P-selectin under flow conditions in vitro. Freshly isolated blood DCs were further observed to roll continuously along noninflamed murine dermal endothelium in vivo. This interaction is strictly dependent on endothelial selectins, as shown by experiments with blocking antibodies and with E- and P-selectin-deficient mice. We hypothesize that DCs in blood are constitutively poised at the interface of blood and skin, ready to extravasate upon induction of inflammation, and we showed that cutaneous inflammation results in a rapid recruitment of DCs from the blood to tissues. We propose that this is an important and previously unappreciated element of immunosurveillance.

Animals↗